Although antimicrobial peptides (AMPs) have highly desirable intrinsic characteristics in their commercial product development as new antimicrobials, the limitations of AMPs from experimental to scale development include the low oral bioavailability, and high production costs due to inadequate in vitro/in vivo gene expression- and low scale. Plectasin has good bactericidal activity against Staphylococcus and Streptococcus, and the selective bactericidal activity greatly reduces the damage to the micro-ecosystem when applied in vivo. However, its expression level was relatively low (748.63 mg/L). In view of these situations, this study will optimize and modify the structure of Plectasin, hoping to obtain candidates with high expression, no/low toxicity, and maintain desirable antibacterial activity. Through sequence alignment, Plectasin was used as a template to introduce the degenerate bases, and the screening library was constructed. After three different levels of screening, the candidate sequence PN7 was obtained, and its total protein yield in the supernatant was 5.53 g/L, with the highest value so far for the variants or constructs from the same ancestor source. PN7 had strong activity against several species of Gram-positive bacteria (MIC value range 1~16 μg/mL). It was relatively stable in various conditions in vitro; in addition, the peptide showed no toxicity to mice for 1 week after intraperitoneal injection. Meanwhile, PN7 kills Staphylococcus aureus ATCC 43300 with a mode of a quicker (>99% S. aureus was killed within 2 h, whereas vancomycin at 2× MIC was 8 h.) and longer PAE period. The findings indicate that PN7 may be a novel promising antimicrobial agent, and this study also provides a model or an example for the design, modification, or reconstruction of novel AMPs and their derivatives.
Keywords: DNA library; Staphylococcus aureus; antimicrobial peptides; degenerate bases; recombination expression.